Method for continuously culturing Ehrlichia and Neorickettsia risticii

ABSTRACT

The present invention relates to a method of culturing bacterial organisms belonging to the family Anaplasmataceae in mammalian embryonic or fetal cells. In particular, the present invention is directed to growth of bacterial organisms belonging to the family Anaplasmataceae including organisms belonging to the  Anaplasma, Ehrlichia  and  Neorickettsia  genera. The bacterial organisms may be cultured in mammalian embryonic or fetal host cells including feline embryonic host cells. Bacterial material cultured according to the methods described herein may be used as the basis for vaccines against diseases associated with the Anaplasmataceae bacteria, or as the basis for diagnostic applications useful for diagnosing diseases associated with the Anaplasmataceae bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. application Ser.No. 12/674,114, filed on Feb. 18, 2010, which is a national stage entryunder 35 U.S.C. §371 of PCT/US2008/076025, filed on Sep. 11, 2008, whichclaims priority to U.S. Provisional Application Nos. 60/971,716, filedon Sep. 12, 2007, 60/971,726, filed on Sep. 12, 2007, and 61/031,428,filed on Feb. 26, 2008, the contents of which are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a method of culturing bacterialorganisms belonging to the family Anaplasmataceae in mammalian embryonicor fetal cells, such as feline embryonic or fetal cells. In particular,the present invention is directed to growth of bacterial organismsbelonging to the family Anaplasmataceae including organisms belonging tothe genera Anaplasma, Ehrlichia, Neorickettsia, and Wolbachieae. Thebacterial organisms may be cultured in mammalian embryonic or fetalcells, such as feline embryonic or fetal host cells. Bacterial materialcultured according to the methods described herein may be used as thebasis for vaccines against diseases associated with the Anaplasmataceaebacteria.

BACKGROUND OF THE INVENTION

The bacteria of the family Anaplasmataceae are obligate intracellularparasites. As such, these microorganisms are often difficult to grow andthe diseases they cause are difficult to diagnose. Because of thedifficulty of growing these microorganisms, large-scale preparation ofvaccine antigens is costly and sometimes impossible. The bacteria of theAnaplasmataceae family are pathogenic agents of vector-transmitteddiseases of human and animals. They are mostly transmitted byinvertebrate vectors such as ticks. Within the family Anaplasmataceae,the microorganisms of the genera Anaplasma, Ehrlichia, Neorickettsia andWolbachieae are causative agents of vector-transmitted diseases and aredifficult to grow, especially on a large-scale. Bacterial speciesbelonging to the genus Rickettsieae are not included within the familyAnaplasmataceae.

Anaplasmosis is a major tick-borne disease of cattle endemic in theUnited States. The causative agent, Anaplasma marginale, invades andmultiplies in erythrocytes of cattle, causing mild to severe anemia.Annual mortality and morbidity due to anaplasmosis impacting beef cattleherds has caused millions of dollars worth of damage due to, forexample; weight loss during acute infections and increased veterinarycosts. See e.g., Palmer in Veterinary Protozoan and HemoparasiteVaccines, J. G. Wright (Ed.) CRC Press Inc., Boca Raton, Fla. 1989.

Anaplasma phagocytophilum causes tick borne fever in sheep, cattle, andbison, and is vecotred by I. ricins. See U.S. Pat. No. 6,284,238, whichis fully incorporated by reference herein. In contrast to A. marginalewhich infects red blood cells, A. phagocytophilum infects granulocytes.

Ehrlichia bacteria are closely related to Anaplasma species. Thosespecies for which a biological vector is known are transmitted by ticks,such as for E. canis. While Anaplasma phagocytophilum prefers to infectgranulocytes in its mammalian host, Ehrlichia canis prefers to infectmononuclear white blood cells. Typically, Anaplasma and Ehrlichia arecontained within membrane-bound vacuoles of their respective host cells.

The first Ehrlichia species recognized was E. canis. It occurs in allareas of the world where the vector tick, Rhipicephalus sanguineus (thebrown dog tick), lives. The disease it causes is sometimes called caninetropical pancytopenia. It is a problem especially in all warm areas ofthe world, e.g., the southern U.S., Central and South America, theMediterranean, and South Asia. Ehrlichia canis may be cultivated in thedog cell line DH82 as well as human-dog hybrid cell lines (See Rikihisa,Y. 1991. Clinical Microbiology Reviews, 4:286). Various strains of E.canis are listed in U.S. Patent Application No. 2006/0188524, which isherein incorporated by reference in its entirety. Ehrlichia chaffeensisis associated with human ehrlichiosis. See Maeda, K. et al., 1987, N.Eng. J. Med. 316:853; Dawson, J. E. et al., 1991, J. Clin. Microbiol.29:2741. E. chaffeensis can also be cultured in DH82 cells.

Neorickettsia bacteria are closely related to Ehrlichia and Anaplasmabacteria. Neorickettsia species include N. risticii and N. sennetsu(both of which were previously classified in the genus Ehrlichia. N.risticii is the causative agent of Potomac horse fever. This disease isknown to occur in North America, France and India. N. risticii may begrown in macrophage-monocyte cell lines such as P388D₁, T-84 and U937.N. sennetsu is the causative agent of Sennetsu erlichiosis of humans. N.sennetsu grows in murine and human cell lines such P388D, L929, andHeLa.

Infections with bacterial organisms of the Anaplasmataceae family may bediagnosed by direct microscopic examination and/or serodiagnosis.Treatment with antibiotics may be effective. Equine vaccines to protectagainst N. risticii are commercially available. See Compendium ofVeterinary Products, 6th ed., Aurora Arrioja, ed., North AmericanCompendiums, Ltd., Port Huron, Mich. (2001). At least one cattle vaccineto protect against anaplasmosis is also commercially available. See Id.However, vaccines have not been manufactured and sold for large scaleprevention of other diseases caused by bacterial organisms such as, forexample, E. canis. Presently, it is believed that there are nocommercial vaccines for Ehrlichia canis.

Efforts have been made to grow certain Rickettsiale organisms in avariety of host cells. U.S. Pat. No. 5,192,679, which is hereinincorporated by reference in its entirety, relates to continuouspropagation of E. canis in a canine monocyte macrophage cell line DH82in an in vitro medium that supports the growth of DH82 cells. U.S.Published Application No. 2005/0202046, which is herein incorporated byreference in its entirety, relates to E. canis vaccines where the E.canis was cultured in DH82 cells. U.S. Pat. No. 5,401,656, which isherein incorporated by reference in its entirety, relates to propagationof E. chaffeensis and E. canis in an immortalized human endothelial cellline. U.S. Pat. No. 5,869,335, which is herein incorporated by referencein its entirety, relates to culturing certain Rickettsiale bacteria inIxodes scapularis cell lines. U.S. Pat. No. 5,989,848, which is hereinincorporated by reference in its entirety, relates to growth of certainEhrlichial species on an immortalized human endothelial cell line. U.S.Pat. No. 3,616,202, which is herein incorporated by reference in itsentirety, relates to growth of Anaplasma marginale in a rabbit bonemarrow tissue culture. U.S. Published Patent Application No.2006/0057699, which is herein incorporated by reference in its entirety,relates to growth of certain Anaplasma species in mammalian cells. U.S.Published Patent Application No. 2003/0003508, which is hereinincorporated by reference in its entirety, relates to culturingRickettsia pulicis bacterium on a Xenopus laevis cell line. U.S. Pat.Nos. 5,955,359 and 5,976,860, which are herein incorporated by referencein their entirety, relate to culturing certain bacterial speciesbelonging to the Rickettsiales order in certain mammalian cell lines.U.S. Pat. No. 5,877,159, which is herein incorporated by reference inits entirety, relates to methods for introducing and expressing genes inanimal cells using certain live invasive bacterial vectors.

A thesis discussing immunization of dogs against canine ehrlichiosisusing inactivated Ehrlichia canis organisms has been submitted. SunitaMahan, Immunisation of German shepherd dogs against canine ehrlichiosisusing inactivated Ehrlichia canis organisms, thesis submitted to theFaculty of Veterinary Science at the University of Zimbabwe (May 1997).This thesis discusses use of a β-propiolactone inactivated E. canisorganisms in combination with Quill A.

Because growth of bacterial species belonging to the Anaplasmataceaefamily in host cells has met with only limited success and apparentlyhas not translated into an abundant supply of vaccines, there remains ageneral need to develop culturing systems for growing such bacterialspecies to facilitate the study of these pathogenic microorganisms andfor the development of vaccines to guard against the diseases theycause. There is also a need to develop a large scale culturing systemfor preparation of large amounts of antigen from such microorganisms foruse in diagnostics and vaccines.

SUMMARY OF THE INVENTION

The present invention broadly relates to culturing bacterial organismsbelonging to the Anaplasmataceae family in mammalian embryonic or fetalhost cells. The cultured bacterial organisms can be used as a vaccineagainst diseases caused by the bacterial organisms. The antigen used inthe vaccine can be made from the bacterial organisms isolated from themammalian host cells. Alternatively, the antigen used in the vaccine canbe made from cultures of the mammalian host cells infected with thebacterial organisms. The bacterial organisms (or the host cell ifpresent) can be inactivated. Alternatively, the bacterial organisms canbe attenuated live such that they replicate within an animal to whichthey are administered one or more times without causing the diseasestate typical of the non-attenuated pathogenic form of the bacterialorganism.

With greater particularity, the present invention relates to culturingbacterial organisms of the Anaplasmataceae family in host cells that areembryonic mammalian cells. In one embodiment of the invention, thebacteria are cultured in non-human embryonic mammalian cells. Such hostcells can be obtained from any part of a non-human animal embryo orfetus. Such host cells can be differentiated or non-differentiated. Theembryonic host cells can be derived from feline, canine, murine, swine,bovine, ovine, simian or equine embryos or fetuses. In one embodiment ofthe invention, the embryonic host cells are derived from feline embryosor fetuses.

In one embodiment of the invention, the bacterial organisms of theAnaplasmataceae family belong to the genera Anaplasma, Ehrlichia orNeorickettsia. Bacterial organisms belonging to the familyAnaplasmataceae do not include those bacterial organisms belonging tothe family Rickettsiaceae. (The Rickettsiaceae family includes the genusRickettsieae, which includes the species R. orientia and R. rickettsia.)The specific bacterial organisms belonging to the Anaplasma genus can beA. bovis, A. centrale, A. marginale, and A. phagocytophilum. Thespecific bacterial organism belonging to the Ehrlichia genus can be E.canis. The specific bacterial organism belonging to the Neorickettsiagenus can be N. risticii.

The present invention relates to a method of culturing a bacterialspecies from the Anaplasmataceae family comprising: i) obtainingbacterial species from the Anaplasmataceae family; ii) infectingnon-human mammalian embryonic cells with said bacterial species; andiii) culturing said non-human mammalian embryonic cells under conditionsconducive to propagating the non-human mammalian embryonic cells,thereby culturing the bacterial species. The bacterial species can beobtained in a purified state free of any host cell, in a state where itis present in a host cell, or in a state where it is present in ahomogenate of infected animal tissue. In one embodiment, the non-humanmammalian embryonic cells are infected with a homogenate of mammaliancells isolated from an animal infected with an Anaplasmataceae organism.In another embodiment, the non-human mammalian embryonic cells areinfected by exposing the embryonic cells to an Anaplasmataceae organism.The Anaplasmataceae bacterial species can be from the genera Anaplasma,Ehrlichia or Neorickettsia. The Anaplasmataceae bacterial species can beAnaplasma bovis, Ehrlichia canis, or Neorickettsia risticii. In oneembodiment, the non-human mammalian embryonic cells are feline cells.The feline cells can be feline embryonic fibroblast cells, FEA felineembryonic cells, or felis catus whole fetus cells. The non-humanmammalian embryonic cells can be undifferentiated and/or immortalized.In another embodiment, the non-human mammalian embryonic cells aremonkey embryo kidney epithelial cells.

The present invention also relates to compositions comprising non-humanmammalian embryonic cells infected with a bacterial species from theAnaplasmataceae family. The bacterial species can be from the generaAnaplasma, Ehrlichia or Neorickettsia. The Anaplasmataceae bacterialspecies can be any known to the skilled artisan including, withoutlimitation, Anaplasma bovis, Anaplasma phagocytophilum, Ehrlichia canis,or Neorickettsia risticii. The non-human embryonic mammalian cells canbe feline embryonic fibroblast (FEF) cells, FEA feline embryonic cells,or felis catus whole fetus cells. The non-human mammalian embryoniccells can be undifferentiated and/or immortalized. The non-humanembryonic mammalian cells can also be monkey embryo kidney epithelialcells.

The present invention also relates to methods of preventing infection ina mammal by administering to the mammal a vaccine based upon materialcultured according to the methods described herein. The presentinvention also pertains to methods of protecting a mammal byadministering to the mammal a vaccine based upon material culturedaccording to the methods described herein. The present invention alsopertains to methods of treating a mammal by administering to the mammala vaccine based upon material cultured according to the methodsdescribed herein. In particular, the present invention relates toprotecting a mammal against a disease caused by an organism belonging tothe family Anaplasmataceae by providing to the mammal a therapeuticallyeffective amount of a Anaplasmataceae bacterial antigen. The mammal canbe a human, monkey, cat, dog, horse, cow, pig, sheep or goat. In oneembodiment of the invention, the animal is a dog.

The present invention also pertains to administering to a mammal animmunologically protective amount of material cultured according to themethods described herein; or administering to a mammal an effectiveamount of material cultured according to the methods described herein toproduce an immune response.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise stated, all terms used herein have their ordinarymeaning as would be understood by the skilled artisan. Terms for whichexplicit definitions are provided below have in addition to theirexplicit meaning the meaning typically ascribed by the ordinarilyskilled artisan.

The present invention relates to methods of culturing microorganisms.More particularly, the present invention is related to methods ofgrowing bacterial organisms belonging to the family Anaplasmataceae.More particularly, the present invention is related to methods ofcontinuously growing organisms belonging to the genera Anaplasma,Ehrlichia or Neorickettsia.

Non-limiting examples of species belonging to the Anaplasma genus thatmay be used according to the present invention include A. bovis, A.centrale, A. marginale, A. ovis, A. platys and A. phagocytophilum(previously referred to as Ehrlichia phagocytophila, Ehrlichia equi,human granulocytic ehrlichiosis agent or HGE agent). Non-limitingexamples of species belonging to the Ehrlichia genus that may be usedaccording to the present invention include E. canis, E. chaffeensis, andE. muris. Non-limiting examples of species belonging to theNeorickettsia genus that may be used according to the present inventioninclude N. helminthoeca, N. risticii (Potomac horse fever, formerlytermed E. risticii), and N. sennetsu.

According to the present invention, bacterial organisms of theAnaplasmataceae family are grown in mammalian embryonic or fetal hostcells. As used herein, “host cells” are cells that bacterial organismsof the Anaplasmataceae family can infect and in which the bacterialorganisms can replicate. Non-limiting examples of mammalian embryonic orfetal host cells include human, feline, canine, murine, swine, bovine,ovine, simian or equine embryonic or fetal cells. As used herein,“infected host cells” refer to host cells that contain one or moreAnaplasmataceae bacteria.

The host cells can be derived from a single cell isolated from mammalianembryonic or fetal tissue. For example, the cells can be derived fromembryonic feline, canine, bovine, equine, murine, swine, simian or humantissue.

In one embodiment of the present invention, bacterial species belongingto the Anaplasmataceae are cultured in host cells that are derived fromnon-human mammalian embryonic or fetal tissue. A non-limiting examplesof feline embryonic or fetal cells that may be used according to thepresent invention include feline embryonic fibroblasts (FEF) cells, FEAfeline embryonic cells (University of Glasgow, Glagow, Scotland; asdescribed in Jarrett, O. et al., J. gen. Prot 20:169-175 (1973)), andfelis catus whole fetus cells (FCWF-4, American Type Culture Collection,P.O. Box 1549 Manassas, Va. 20108, United States (hereafter, “ATCC”)deposit CRL-2787).

The bacterial organisms cultured according to the present invention maybe used for vaccines, diagnostics or further research including, forexample, production of quantities of biological molecules for isolationand study. Accordingly, the bacteria cultured according to presentinvention may be formulated in a vaccine for administration to a mammalto prevent infection or ameliorate disease caused by the bacterialorganism. For example, the bacteria cultured in the host cells could beEhrlichia canis for use in a vaccine to be given to canines to preventor ameliorate canine ehrlichiosis.

Hence, the present invention also relates to immunogenic compositions orvaccines based upon material cultured according to the presentinvention. The therapeutic agent (also referred to as the antigen,active agent, or the immunogenic composition) that can serve as thebasis for a vaccine can be one or more of the following:

a) harvested cultures of host cells that are infected withAnaplasmataceae bacteria;

b) extracts or fractions of (a) that are enhanced with respect to theconcentration of the Anaplasmataceae bacteria contained within theinfected host cells;

c) Anaplasmataceae bacteria enhanced extracts of (a) that containremnants of the host cells;

d) Anaplasmataceae bacteria extracts of (a) that do not contain remnantsof the host cells; or

e) isolated Anaplasmataceae bacterial immunogens.

“Isolated” when used herein means removed from its naturally occurringenvironment. Hence, isolated Anaplasmataceae bacterial cells broadlyinclude those which have been removed from their naturally occurringenvironments, which environments can include arthropods, insects orwhole live or dead infected mammals. Isolated Anaplasmataceae bacterialcells include thus; that, are contained within mammalian tissue that hasbeen removed from a Anaplasmataceae bacteria-infected mammal. IsolatedAnaplasmataceae bacterial cells also include those that are completelyor partially separated from mammalian cells of a Anaplasmataceaebacteria-infected mammal, such as from lysing the host cells. IsolatedAnaplasmataceae bacterial cells also include those that are containedwithin host cells as described herein, or separated partially orcompletely therefrom. Isolated Anaplasmataceae bacterial cells alsoinclude those that are substantially free of other microorganisms, e.g.,in a culture.

“Isolated” as used in “isolated Anaplasmataceae bacterial immunogens”refers to bacterial immunogens that have been completely or partiallyseparated from their respective source Anaplasmataceae bacteria.Compositions of isolated Anaplasmataceae bacterial immunogens caninclude some whole intact Anaplasmataceae bacteria, portions orcomponents of Anaplasmataceae bacteria, whole intact host cell, and/orportions or components of host cells. Isolated Anaplasmataceae bacterialimmunogens also include compositions that are enriched with respect toone or more Anaplasmataceae bacterial biomolecules.

“Anaplasmataceae bacterial immunogens” as used herein includes wholeAnaplasmataceae bacteria that are inactivated or modified live bacteria.Anaplasmataceae bacterial immunogen as used herein can also includeproteins (lipoproteins, membranous proteins, cytosolic proteins),immunogenic fragments of such proteins, nucleic acids, lipids,saccharides, lipopolysaccharides or other biological molecules derivedfrom the Anaplasmataceae bacteria. Anaplasmataceae bacterial immunogencan be whole Anaplasmataceae bacterial cells or parts thereof that arepresent in host cells, wherein both the bacterial and host cells arekilled or inactivated. Anaplasmataceae bacterial immunogen can also bewhole Anaplasmataceae bacterial cells or parts thereof that are presentin host cells, wherein the bacterial or host cells are not killed orinactivated.

The skilled artisan is generally familiar with the techniques by whichbacterial or host cells can be killed or inactivated. Such techniquesinclude, physical, chemical and biological means. Non-limiting examplesof inactivation techniques include sonication, freeze-thaw techniques,pressure, treatment with heat, chemicals or enzymes. Non-limitingexamples of chemical inactivation agents include treatment with binaryethyleneamine (BEA) and formalin (formaldehyde solution).

As stated above, the material cultured according to the presentinvention can be used to make antigen for vaccines. As used herein, theterm “vaccine(s)” means and refers to a product, the administration ofwhich is intended to elicit an immune response that can prevent and/orlessen the severity of one or more infectious diseases. A vaccinecontains an antigen (or, “active agent,” “immunogen,” “therapeuticagent,” or “immunogenic composition”) that may be material culturedaccording to the present invention including a host cell infected withAnaplasmataceae bacteria, whole intact Anaplasmataceae bacteria, orbacterial fractions or parts or biomolecules of a Anaplasmataceaebacteria that act to stimulate the immune system in an animal. Anantigen may be a live attenuated or killed preparation ofAnaplasmataceae bacteria-infected host cells, live attenuated or killedAnaplasmataceae bacteria, living irradiated cells, crude fractions orpurified Anaplasmataceae bacterial immunogens. Hence, a vaccine cancomprise enriched, isolated or purified antigen. The vaccines can bemade from inactivated or killed cultures of Anaplasmataceae infectedhost cells, or inactivated or killed Anaplasmataceae bacteria.

A vaccine may also comprise a combination of antigens from more than oneAnaplasmataceae bacterial species or from other pathogens (e.g. viral,bacterial parasitical or fungal) as described further below.

Vaccines made from material cultured according to the present inventioncomprise a therapeutically effective amount of the antigen. In thecontext of this disclosure, a “therapeutically effective amount” refersto an amount of an antigen or vaccine that would induce an immuneresponse in a mammal receiving the antigen or vaccine which is adequateto prevent or ameliorate signs or symptoms of disease, including adversehealth effects or complications thereof, caused by infection with apathogenic Anaplasmataceae bacterium. Humoral immunity or cell-mediatedimmunity or both humoral and cell-mediated immunity may be induced. Theimmunogenic response of an animal to a vaccine may be evaluated, e.g.,indirectly through measurement of antibody titers, via microscopicanalysis, or directly through monitoring signs and symptoms afterchallenge with wild type strain. The protective immunity conferred by avaccine may be evaluated by measuring, e.g., reduction in clinical signssuch as mortality, morbidity, body temperature and overall physicalcondition and overall health and performance of the subject. The amountof a vaccine that is therapeutically effective may vary depending on theparticular virus used, or the condition of the subject, and may bedetermined by one skilled in the art.

The material cultured according to the present invention can also beused to make immunogenic compositions that stimulate an immune responsein a subject mammal to which the compositions are administered. Suchcompositions can be used to identify antigens that can serve as thebasis of a vaccine. Thus, for example, immunogenic compositionscomprising material cultured according to, the present invention can beadministered to a subject mammal. Thereafter, the antibody titer of thesubject mammal can be monitored and candidate Anaplasmataceae bacterialantigens can be selected for use or further study in vaccines.Immunoactive compositions of the present invention include compositionsthat stimulate a humoral immune response and/or a cell-mediated immuneresponse in the subject receiving a vaccine.

As used herein, an “immune response” refers to the subject mammal'sactive immunity response due to having received one or more vaccinesbased upon material cultured according to the methods described herein.The immune response can include the production of one or more antibodyin response to the antigen or immunogen present in the vaccine. “Immuneresponse” in a subject refers to the development of a humoral immuneresponse, a cellular immune response, or a humoral and a cellular immuneresponse to an antigen. Immune responses may be determined usingstandard immunoassays and neutralization assays, which are known in theart.

Vaccines made from material cultured according to the present inventioncan be used to prevent infection within a subject mammal, protect asubject mammal, or treat a subject mammal.

“Preventing infection” and like terms means to prevent or inhibit thereplication of the bacteria which cause the identified disease, toinhibit transmission of the bacteria or virus, or to prevent thebacteria from establishing itself in its host animal, or to alleviatethe symptoms of the disease caused by infection. The treatment isconsidered therapeutic if there is a reduction in bacterial load.

“Protection”, “Protecting”, and the like, as used herein with respect toa bacteria, means that the vaccine prevents or reduces the symptoms ofthe disease caused by the organism from which the antigen(s) used in thevaccine is derived. The terms “protection” and “protecting” and thelike, also mean that the vaccine may be used to “treat” the disease orone of more symptoms of the disease that already exists in a subject.

“Treating” refers to reversing, alleviating, inhibiting the progress of,or preventing a disorder, condition or disease to which such termapplies, or to preventing one or more symptoms of such disorder,condition or disease. Treating also refers to accelerating the recoveryfrom an infection by one or more Anaplasmataceae organisms. “Treatment”refers to the act of “treating”.

Hence, vaccines made from material cultured according to the presentinvention can be used to prevent Anaplasmataceae bacterial infection ina subject mammal, protect a subject mammal against Anaplasmataceaebacteria, and treat a subject mammal for Anaplasmataceae bacterialinfection. Such prevention, protection or treatment can include (withoutlimitation) reducing or eliminating the risk of infection by thepathogenic Anaplasmataceae organism, ameliorating or alleviating thesymptoms of an infection by such Anaplasmataceae organism, reduction inAnaplasmataceae bacterial load, decreasing incidence or duration ofAnaplasmataceae infections, reducing acute phase serum protein levels ofAnaplasmataceae bacteria, reduced rectal temperatures, and/or increasein food uptake and/or growth, for example.

“Pharmaceutically acceptable” as used herein refers to substances (e.g.,adjuvants, immunostimulants, carriers, diluents, emulsifying orstabilizing agents), which are within the scope of sound medicaljudgment, suitable for use in contact with the tissues of subjectswithout undue toxicity, irritation, allergic response, and the Like,commensurate with a reasonable benefit-to-risk ratio, and effective fortheir intended use. Pharmaceutically acceptable substances do notinterfere with the effectiveness of the therapeutic agent and are nottoxic to the subject to whom it is administered.

“Subject” or “subject mammal” refers to any animal having an immunesystem, which includes mammals such as humans, cats, cattle, horses,swine, and dogs.

Material cultured according to the present invention can also be used indiagnostic applications to diagnose the presence of diseases orillnesses caused by Anaplasmataceae bacteria. Non-limiting examples ofsuch diagnostic applications include use of bacterial fractions,proteins or other biomolecules in antibody binding assays. The bacterialfractions, proteins or other biomolecules may also be used to generatepolyclonal or monoclonal antibodies for such assays.

Host Cell Growth

Host cells for culturing bacterial organisms according to the presentinvention are first prepared prior to infecting with the desiredbacterial organism. A sample of an isolated feline embryonic cell lineis seeded into media for either suspended or adherent growth. As usedherein, adherent growth conditions wherein a layer of cells coatssurfaces contained within the vesicle in which the cells are cultured.The surfaces can include the interior surface of the vesicle itself, orsurfaces of glass or polymeric beads contained within the vesicle toincrease surface area. Microcarriers can also be used to increasesurface area and host cell growth. In contrast to adherent growth, itmay be possible to grow the host cells in suspension, in which the hostcells need not bind to surfaces within the culturing vesicle.

The skilled artisan is generally familiar with the varieties ofculturing media that may be used to grow up the host cells. The hostcell growth media may be derived from animals. Alternatively, the hostcell growth media may be vegetable or yeast based, and may be animalprotein-free. The growth media may be derived from soy bean extracts orfrom other protein-rich plants or protein-rich plant food productsincluding, for example, legumes. Non-limiting example of specific mediauseful for growing host cells include Eagle's Minimal Essential Media(MEM), Glasgow-Minimal Essential Media, RPMI1640, OptiMEM, AIM V.

The growth media can contain or be supplemented with fetal bovine serum(FBS), tryptose solution, lacto-albumin hydrosolate solution,L-glutamine, sodium bicarbonate; lactalbumin hydrolysate, Polymyxin B,sodium pyruvate, glucose, magnesium sulfate.

Fresh growth media can be refed or replenished to the host cells priorto or after infection or exposure of the host cells to theAnaplasmataceae bacteria.

Cells can be grown at 36-38° C. for 2-9 days at 5% CO₂.

Infecting the Host Cells

The host cells may be exposed to or infected with bacterial organisms ofthe Anaplasmataceae family by bringing the host cells into contact withother eukaryotic cells known to be infected with the bacterialorganisms. The skilled artisan is familiar with determining whether suchother eukaryotic cells from a mammal, for example, are infected withsuch bacterial organisms. The infected mammalian cells may be derivedfrom any tissue, including the spleen, liver, pancreas, lungs, heart orother muscle tissue, brain, gall bladder, blood, kidneys, lymph nodes orstomach. The infected mammalian cells may be prepared from a tissueextract via blender homogenization in an appropriate isotonic solution.The homogenate can then be used to innoculate (i.e., infect) a cultureof host cells, applied as a layer over the host cells or simply broughtinto contact with them.

Alternatively, the host cells may be exposed to or infected withisolated bacterial organisms of the Anaplasmataceae family. The skilledartisan is familiar with techniques of isolating such bacterialorganisms, or can obtain stocks of isolated bacterial organisms from abiological depository.

The growth medium used to prepare host cells prior to contact withAnaplasmataceae bacteria may be the same as the medium used to propagatethe host cells after such contact. The Anaplasmataceae bacteria-exposed(or infected) host cells may be cultured for up to 95 days, up to 35days, or for about 5 to 10 days, to achieve a titer of ≧1×10⁴ TCID₅₀(Tissue Culture Infectious Dose), and then the culture may be harvestedand processed.

Harvesting

The Anaplasmataceae bacterial infected host cells may be harvested bycollecting the tissue cell culture fluids and/or cells. The host cellsmay be harvested from the media (and the culture vesicles) with theAnaplasmataceae bacterial cells contained with the walls of the hostcells. Alternatively, during harvesting the concentration of theAnaplasmataceae bacteria may be enriched by techniques that improve theliberation of the infective bacterial cells from the growth substrate,e.g. sonication, freeze thawing, heating or chemical or selectiveenzymatic lysis of the eukaryotic host cells. An enriched harvest ofAnaplasmataceae bacteria can include material that is free of host cellsor host cell material. Alternatively, an enriched harvest ofAnaplasmataceae bacteria can include material that contains host cellsor host cell material.

Inactivating

The skilled artisan is generally familiar with the techniques by whichbacterial or host cells can be killed or inactivated. Such techniquesinclude, physical, chemical and biological means. Non-limiting examplesof inactivation techniques include sonication, freeze-thaw techniques,pressure, treatment with heat, chemicals or enzymes. Non-limitingexamples of chemical inactivation agents include treatment with binaryethyleneimine (BEI), formalin (formaldehyde solution),beta-propiolactone, merthiolate, gluteraldehyde, sodium dodecyl sulfate,or the like, or a mixture thereof. The host cells can also beinactivated by heat or psoralen in the presence of ultraviolet light.These chemical inactivation agents or physical inactivation means canalso be used to inactivate the Anaplasmataceae bacterial cells aftertheir having been extracted or separated from the host cells.

Formulating

The inactivated, infected host cells or enriched Anaplasmataceaebacterial cells can serve as the antigen and may be formulated as aliquid suspension or may be lyophilized for its use in the preparationof a vaccine against diseases caused by Anaplasmataceae organisms.Material cultured according to the present invention can be formulatedwith any pharmaceutically acceptable adjuvants, immunostimulants,carriers, diluents, emulsifying or stabilizing agents, non-limitingexamples of which are discussed below. The skilled artisan, however,would recognize that other adjuvants, immunostimulants, carriers,diluents, emulsifying agents or stabilizing agents may be used informulating vaccines based upon material cultured according to thepresent invention.

Adjuvants & Immunostimulants

An adjuvant in general is a substance that boosts the immune response ofthe target in a non-specific manner. Many different adjuvants are knownin the art. Non-limiting examples of adjuvants that may be used in theformulation of a vaccine made with material cultured according to thepresent invention include aluminum salts (e.g., alum, aluminumhydroxide, aluminum phosphate, aluminum oxide), cholesterol,monophosphoryl lipid A adjuvants, amphigen, tocophenols, monophosphenyllipid A, muramyl dipeptide, oil emulsions, glucans, carbomers, blockcopolymers, Avridine lipid-amine adjuvant, heat-labile enterotoxin fromE. coli (recombinant or otherwise), cholera toxin, or muramyl dipeptide,Freund's Complete and-Incomplete adjuvant, vitamin E, non-ionic blockpolymers and polyamines such as dextransulphate, carbopol, pyran,saponins and saponin derivatives, block co-polymers, and adjuvants suchas those identified in U.S. Pat. Nos. 4,578,269, 4,744,983, 5,254,339,which are all herein fully incorporated by reference. Non-limitingexamples of peptides that can serve as adjuvants includemuramyldipeptides, dimethylglycine, or tuftsin. Non-limiting examples ofoils that can serve as adjuvants include mineral oil, vegetable oils oremulsions thereof.

Vaccines made from material cultured according to the present inventionmay be formulated as an oil-in water emulsions or as a water-in-oilemulsions. Non-limiting examples of oil-in-water emulsions includeparaffin oil-in-water emulsions, or emulsions made from one or more ofsqualene, block copolymers of ethylene oxide and propylene oxide,polysorbate surfactants, and/or threonyl analogs of muramyl dipeptide.

Oils used as adjuvants may be metabolizable by the subject receiving thevaccine such as vegetable or animal oils. Such oils typically consistlargely of mixtures of triacylglycerols, also known as triglycerides orneutral fats. These nonpolar, water insoluble substances are fatty acidtriesters of glycerol. Triacylglycerols differ according to the identityand placement of their three fatty acid residues.

Adjuvants can also be non-metabolizable consisting of components thatcannot be metabolized by the body of the animal subject to which theemulsion is administered. Non-metabolizable oils suitable for use in theemulsions of the present invention include alkanes, alkenes, alkynes,and their corresponding acids and alcohols, the ethers and estersthereof, and mixtures thereof. The individual compounds of the oil maybe light hydrocarbon compounds, e.g., compounds having 6 to 30 carbonatoms. The oil may be synthetically prepared or purified from petroleumproducts. Non-limiting examples of non-metabolizable oils for use in thepreparation of vaccines based upon material cultured according to thepresent invention include mineral oil, paraffin oil, and cycloparaffins,for example. The term “mineral oil” refers to a non-metabolizableadjuvant oil that is a mixture of liquid hydrocarbons obtained frompetrolatum via a distillation technique. The term is synonymous with“liquefied paraffin”, “liquid petrolatum” and “white mineral oil.” Theterm is also intended to include “light mineral oil,” i.e., oil which issimilarly obtained by distillation of petrolatum, but which has aslightly lower specific gravity than white mineral oil.

Other compounds capable of enhancing a humoral immunity response thatmay be used in the formulation of vaccines based upon material culturedaccording to the present invention include, without limitation, ethylenemaleic anhydrate (EMA) copolymer, latex emulsions of a copolymer ofstyrene with a mixture of acrylic acid and methacrylic acid.

In addition to the adjuvant, a vaccine based upon material culturedaccording to the present invention can include immunomodulatory agentssuch as, e.g., interleukins, interferons, or other cytokines (e.g.,Th1-related cytokines, such as interleukin-12 (IL-12), interleukin-18(IL-18), or gamma interferon).

The amount of adjuvant or immunostimulant added in a vaccine formulationbased upon material cultured according to the present invention dependson the nature of the adjuvant or immunostimulant itself. The skilledartisan is capable of selecting an amount that is sufficient to enhancean immune response to the Anaplasmataceae bacterial immunizing agent.

Carriers

Pharmaceutically acceptable carriers suitable for use in vaccineformulated based upon material cultured according to the presentinvention may be any conventional liquid carrier suitable for veterinarypharmaceutical compositions, including balanced salt solutions such asare suitable for use in tissue culture media. Pharmaceuticallyacceptable carriers are understood to be compounds that do not adverselyeffect the health of the animal to be vaccinated, at least not to theextent that the adverse effect is worse than the effects seen when theanimal is not vaccinated. Suitable carriers also include sterile water,saline, aqueous buffers such as PBS, solvents, diluents, isotonicagents, buffering agents, dextrose, ethanol, mannitol, sorbitol, lactoseand glycerol, and the like.

Vehicle

Vaccines formulated from material cultured according to the presentinvention can also comprise a vehicle. A vehicle is a compound to whichthe host cells, Anaplasmataceae bacterial cells, or proteins, proteinfragments, nucleic acids or parts thereof, adhere, without beingcovalently bound to it. Non-limiting examples of such vehicles includebio-microcapsules, micro-alginates, liposomes and macrosols. Somematerials that serve as adjuvants can also serve as vehicles such asaluminum-hydroxide, aluminum phosphate, aluminum sulphate or aluminumoxide, silica, kaolin, and bentonite, all known in the art.

Stabilizers

Often, a vaccine is mixed with stabilizers, e.g. to protectdegradation-prone components from being degraded, to enhance theshelf-life of the vaccine, or to improve freeze-drying efficiency.Non-limiting examples of stabilizers that may be added to vaccineformulations based upon material cultured according to the presentinvention include SPGA (Bovarnik et al., 1950, J. Bacteriology, vol. 59,p. 509), skimmed milk, gelatins, bovine serum albumin, carbohydrates(e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran orglucose), proteins (e.g., albumin, casein or degradation productsthereof), non-animal origin stabilizers, and buffers (e.g. alkali metalphosphates). In lyophilized vaccine compositions, one or morestabilizers can be added.

Multivalent Vaccines

The immunogen harvested from the material cultured according to thepresent invention may be formulated in a vaccine comprising one or moreadditional immunogens. The additional immunoactive component(s) may bewhole parasite, bacteria or virus (inactivated or modified live), or afractionated portion or extract thereof (e.g., proteins, lipids,lipopolysacharide, carbohydrate or nucleic acid).

Where the immunogen harvested from the material cultured according tothe present invention is used in a canine vaccine, antigens for othercanine pathogens may be added into the formulation. Non-limitingexamples of other pathogens for which additional antigens may be addedinclude Bordetella bronchiseptica, canine distemper virus (CDV), canineadenovirus types 1 and 2 (CAV-1, CAV-2), canine parainfluenza (CPI)virus, canine coronavirus (CCV), canine parvovirus (CPV), Leptospirainterrogans serovar canicola, Leptospira interrogans serovaricterohaemorrhagiae, Leptospira interrogans serovar bratislava,Leptospira interrogans serovar pomona; Leptospira kirschneri serovargrippotyphosa, rabies virus, Borrelia burgdorferi, canine rotavirus(CRV), canine herpesvirus (CHV), and Minute Virus of Canines (MVC),Babesia canis, Giardia and Leishmania.

Alternatively, a vaccine based upon material cultured according to thepresent invention may be administered simultaneously or concomitantlywith other live or inactivated vaccines.

Freeze-Drying/Reconstitution

For reasons of stability or economy, vaccines based upon materialcultured according to the present invention may be freeze-dried. Ingeneral this will enable prolonged storage at temperatures above 0° C.,e.g. at 4° C. Procedures for freeze-drying are known to persons skilledin the art; equipment for freeze-drying at different scales is availablecommercially. To reconstitute the freeze-dried vaccine, it may besuspended in a physiologically acceptable diluent. Such diluents may beas simple as sterile water, or a physiological salt solution or othercarrier as discussed above.

Dosaging

Vaccines based upon material cultured according to the present inventionmay be formulated in a dosage unit form to facilitate administration andensure uniformity of dosage. Herein, a dosage unit as it pertains to thevaccine composition refers to physically discrete units suitable asunitary dosages for animals, each unit containing a predeterminedquantity of Anaplasmataceae bacterial immunogen calculated to producethe desired immunogenic effect in association with the required adjuvantsystem and carrier or vehicle.

The effective immunizing amount of Anaplasmataceae bacterial immunogencan vary depending upon the chosen strain or strains and may be anyamount sufficient to evoke a protective immune response. For example,amounts wherein the dosage unit comprises at least about 1×10⁴ TCID₅₀inactivated Anaplasmataceae bacterin are suitable.

Administering

Administration of the vaccine to a subject results in stimulating animmune response in the subject mammal. The route of administration forvaccines based upon material cultured according to the present inventionmay be administered to the mammalian target according to methods knownin the art. Such methods include, but are not limited to, intradermal,intramuscular, intraocular, intraperitoneal, intravenous, oral,oronasal, and subcutaneous, as well as inhalation, suppository, ortransdermal. Routes of administration include intradermal,intramuscular, intraperitoneal, oronasal, and subcutaneous injection.The vaccine may be administered by any means that includes, but is notlimited to, syringes, nebulizers, misters, needleless injection devices,or microprojectile bombardment gene guns (Biolistic bombardment).

Alternative routes of application that are feasible are by topicalapplication as a drop, spray, gel or ointment to the mucosal epitheliumof the eye, nose, mouth, anus, or vagina, or onto the epidermis of theouter skin at any part of the body; by spray as aerosol, or powder.Alternatively, application may be via the alimentary route, by combiningwith the food, feed or drinking water e.g. as a powder, a liquid, ortablet, or by administration directly into the mouth as a liquid, a gel,a tablet, or a capsule, or to the anus as a suppository. The preferredapplication route is by intramuscular or by subcutaneous injection.

The vaccine according to the invention may be in several forms, e.g.: aliquid, a gel, an ointment, a powder, a tablet, or a capsule, dependingon the desired method of application to the target.

The scheme of the application of the vaccine according to the inventionto the target mammalian may be in single or multiple doses, which may begiven at the same time or sequentially, in a manner compatible with thedosage and formulation, and in such an amount as will be immunologicallyeffective.

Challenge Model

In order to effectively study and evaluate the pathogenic mechanisms ofthe Anaplasmataceae bacteria and the defense mechanisms of the hostmammals and thereby to advance the vaccine art and improve vaccineproducts, an effective challenge model should be employed.

A challenge model for canine ehrlichiosis, for example, may be basedupon the percentage of test animals to demonstrate persistent and severeclinical symptoms that are commonly associated with canine ehrlichiosis,such as fever, thrombocytopenia, mucopurulent ocular discharge,dehydration, or the like. Alternatively, the challenge model describedby published U.S. Application No. 2006/0188524 (which is herein whollyincorporated by reference) may be employed. This E. canis challenge maybe obtained in a test animal by administering to said test animal achallenge stock of peripheral blood mononuclear cells (PBMC) containinga virulent culture of live E. canis bacteria. The virulent E. canisculture is prepared by repeatedly passaging the E. canis microorganismsuch as E. canis Ebony, E. canis Broadfoot or the like, in a host;separating the PBMC from the host blood sample; and mixing the separatedPBMC with 20% fetal bovine serum and 10% dimethyl sulfoxide.

A method for the induction of clinical canine ehrlichiosis in a testanimal includes administering to said animal an effective amount of anE. canis challenge stock, consisting essentially of a virulent E. canismicroorganism in peripheral blood mononuclear cells. Viable cultures ofeach of E. canis Broadfoot (sometimes referred to as E. canis BF, orBroadfoot), and E. canis Ebony (sometimes referred to as Ebony) havebeen deposited (Feb. 11, 2004) with the ATCC, 10801 UniversityBoulevard, Manassas, Va. 20110-2209 U.S.A., and have been respectivelygiven the ATCC accession numbers PTA-5811 for the Broadfoot strain, andPTA-5812 for the Ebony strain.

Several other cellular diagnostic methods exist to determine thepresence of infection. For example, the presence of infection may bedetermined by direct immunofluorescence. Other methods to detectinfection include staining, e.g., Giemsa, Wright/Giemsa. Acridine Orangecan also be utilized to stain the organisms.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are hereby wholly incorporated by reference.

For a more clear understanding of the invention, the following examplesare set forth below. These examples are merely illustrative and are notunderstood to limit the scope or underlying principles of the inventionin any way. Indeed, various modifications of the invention, in additionto those shown and described herein, will become apparent to thoseskilled in the art from the examples set forth hereinbelow and theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

EXAMPLES Example 1 Growth of E. canis on FEF Cells in the Presence ofDH82 Cells

1.1 Propagation of Uninfected DH82 Cells

One frozen vial of uninfected DH82 cells (American Type CultureCollection (ATCC) accession no. CRL-10389, P.O. Box 1549, Manassas, Va.20108) was thawed, clarified, and used to inoculate a 75-cm² cellculture flask containing DH82 Growth Medium, and incubated at 37° C.with 5% CO₂. DH82 Growth Medium consists of Dulbecco's MEM basesupplemented with 10% fetal bovine serum (FBS) and 1% HEPES. Uponformation of a monolayer, the cells were scraped into the growth medium,harvested, and centrifuged at 1,500 rpm for 10 min. The cell pellet wasresuspended in 5 ml of fresh DH82 Growth Medium and split at a ratioranging from 1:3 to 1:5.

1.2 Infection of Uninfected DH82 Cells with E. canis-Infected DH82 Cells

One frozen vial of E. canis-infected DH82 cells (ATCC accession no.CRL-10390) was thawed, clarified, and used to inoculate a 175-cm² cellculture flask containing a monolayer (80-90% confluent) withapproximately 10⁷ uninfected DH82 cells in DH82 Growth Medium. E.canis-infected DH82 cultures were maintained by refeeding, i.e.,replacing 50% of the spent culture medium with fresh DH82 Growth Mediumas described above. E. canis-infected cultures were monitored by usingeither the Diff-Quik staining method on slides containing acetone-fixedcells according to the manufacturer's directions (VWR, West Chester, Pa.#47733-150) or a standard immunofluorescent antibody (IFA) technique.Briefly, plates containing acetone-fixed cells from E. canis-infectedand uninfected DH82 cultures were incubated with a polyclonal E. canisdog serum, washed with PBS, incubated with fluorescein-labeled goatanti-dog IgG gamma (Kirkegaard and Perry #02-19-02), washed with PBS,and examined with a fluorescence microscope.

1.3 Propagation of Uninfected FEF Cells

One vial of frozen feline embryonic fibroblast (FEF) cells was thawed,clarified, and used to inoculate a 175-cm² cell culture flask containingFEF Growth Medium, and incubated at 37° C. with 5% CO₂. FEF GrowthMedium consists of M6B8 medium (MEM base, Glasgow-MEM base, tryptosephosphate, tryptose, lacto-albumin hydrosolate, L-glutamine, and sodiumbicarbonate) and 5% FBS. After incubation for 4-5 days, the cultureswere ready for passage when the monolayer was 90-95% confluent. Aftertreatment with 0.25% trypsin, the cells were split at a ratio of 1:5 to1:10.

1.4 Infection of Uninfected FEF Cells with E. canis-infected DH82 Cells

E. canis-infected DH82 cells were harvested by scraping into the culturemedium, centrifuged at 1,500 rpm for 10 min, and resuspended at 1×10⁶cells/ml in fresh DH82 Growth Medium. Uninfected FEF cultures that wereseeded at 6×10⁶ cells per 175-cm² cell culture flask in FEF GrowthMedium and incubated for 18-24 hrs at 37° C. with 5% CO₂ were suspendedin culture medium and placed in 75-cm² cell culture flasks at a splitratio of 1:3. Six ml of resuspended E. canis-infected DH82 cells werethen used to infect each 75-cm² cell culture flask containing uninfectedFEF cells in suspension, and incubated at 37° C. with 5%. CO₂. The E.canis-infected DH82/FEF mixed cultures were fed every three days byreplacing spent culture medium with fresh FEF Growth Medium. At 14 dayspost-infection, the mix cultures were split 1:2 and cell suspensionswere transferred into the wells of 24-well cell culture plates at 1 mlper well. Following incubation for 16 hrs at 37° C. with 5% CO₂, culturesupernatant from the wells were transferred onto slides, fixed withacetone, and evaluated for presence of E. canis infection using IFA asdescribed above.

Example 2 Growth of E. canis on a Homogenous Population of FEF Cells

Dogs were infected intravenously with 0.5-2 mL of E. canis-infected DH82cells expanded from cells obtained from the ATCC as described above.Such E. canis-infected DH82 cells are described in U.S. Pat. No.5,192,679, which is fully incorporated by reference herein. Dogs werepositively identified as being infected with E. canis via PCR of spleenand blood DNA. DNA was purified from blood and tissue samples using aQIAamp DNA Mini Kit (Qiagen, Valencia, Calif.) according to themanufacturer's instructions. PCR was performed on a RoboCycle® roboticthermocycler (Stratagene, Cedar Creek, Tex.) using 25 μl reactionsconsisting of 2.5 μl of 10× reaction buffer (Genscript Corporation,Piscataway, N.J.), 0.2 μl of 100 mM dNTPs (Invitrogen Corp., Carlsbad,Calif.), 1 μl of 10 μM oligonucleotide primer 1 (5′-AGA ACG AAC GCT GGCGGC AAG C-3″) and oligonucleotide primer 2 (5′-CGT ATT ACC GCG GCT GCTGGC A-3′), and 0.2 μl of 5 U/μl Taq polymerase (Genscript Corp.) in athermocycling protocol consisting of a preliminary denaturation step of94° C. for 5 min, followed by 35 cycles of 94° C. for 1 min, 60° C. for1 min, and 72° C. for 1 min, followed by a final elongation step of 72°C. for 10 minutes. Homogenates in fresh growth medium were prepared fromsamples of spleens, lymph nodes, or peripheral blood mononuclear cells(PBMCs) obtained from E. canis-infected dogs and used as an overlay toinfect FEF cells.

E. canis-infected spleen homogenate was inoculated into uninfected FEFcells in two separate 75-cm² cell culture flasks containing 30 ml of FEFcell suspension seeded at 2×10⁵ cells/ml per flask bringing thehomogenate to a final dilution of 1:10 to 1:100. Following 18-24 hoursincubation at 37° C. with 5% CO₂, the culture medium was replaced with30 mL of fresh FEF Growth Medium. After 5-7 days of incubation, the cellmonolayer was trypsinized and resuspended in 5-10 nil of fresh FEFGrowth Medium. Five mL of this suspension was then inoculated into eachof two 175-cm² cell culture flasks containing 50 mL of FEF GrowthMedium. Cells were then incubated for 7-10 days at 37° C. with 5% CO₂.To maintain viability, the cultures required “feeding”, which wasaccomplished by replacing 50% of spent culture medium with fresh FEFCulture Medium. After 10-14 more days of incubation at 37° C. with 5%CO₂, the cells were trypsinized and resuspended as described above. Tomaintain continuous propagation, 1 to 2 mL of infected FEF cellsuspension was passed onto uninfected FEF cells and incubated at 37° C.with 5% CO₂. Infected cultures were passed 7 to 13 times usingincubation times ranging from 4 to 14 days. The presence of E. canis inthe cultured FEF cells was confirmed by the use of IFA and PCR asdescribed above.

Example 3 Growth of E. canis on a Homogenous Population of FCWF-4 Cells

3.1 Propagation of Uninfected FCWF-4 Cells (ATCC #CRL-2787)

One frozen vial of uninfected felis catus whoe fetus-4 (FCWF-4) cells(ATCC accession no. CRL-2787) was thawed, clarified, and used toinoculate a 75-cm² cell culture flask containing FCWF Growth Medium, andincubated at 37° C. with 5% CO₂. FCWF Growth Medium consists of E-MEM(Eagle's Minimal Essential medium with Earle's balanced salt solutionand 2 mM L-glutamine), 1.0 mM sodium pyruvate, 0.1 mM nonessential aminoacids, 1.5 g/liter sodium bicarbonate, and 10% FBS. After 4-5 days ofincubation, the 90-95% confluent monolayer was treated with 0.25%trypsin and split at a ratio of 1:4 to 1:6.

3.2 Preparation of Homogenous Population of E. canis-infected FCWF-4Cells

Prior to infection with E. canis, uninfected FCWF-4 cells were seededinto a 175-cm² flask at 6×10⁶ cells per flask and incubated for 18-24hrs. E. canis-infected spleen homogenate was used to infect FCWF-4 cellsas described for FEF cells except the FCWF Growth Medium was used inplace for the FEF Growth Medium. The presence of E. canis in thecultured FCWF-4 cells was confirmed by the use of IFA and PCR asdescribed above

Example 4 Growth of E. Marls on a Homogenous Population of FEA Cells

4.1 Propagation of Uninfected FEA Feline Embryonic Cells

One vial of frozen uninfected FEA feline embryonic cells was thawed,clarified and used to inoculate a 75 cm² flask containing FEA GrowthMedium, and incubated at 37° C. with 5% CO2. FEA Growth Medium consistsof Dulbeccos MEM, 2 mM L-glutamine, 1.0 mM sodium pyruvate and 10% FBS.After 7 days of incubation, the confluent monolayer was treated with0.25% trypsin and passed at a split ratio of 1:2.

4.2 Preparation of Homogenous Population of E. Muris-infected FEA FelineEmbryonic Cells

Uninfected DH82 cells were propagated as described above, and infectedwith E. muris using E. muris-infected DH82 cells (ATCC accession no.VR-1411—Asuke strain). The protocol for the preparation of materials andinfection essentially followed the protocol as described above for E.canis-infected DH82 cells, except that E. muris-infected DH82 cells weresubstituted for E. canis-infected DH82 cells. Mice were infectedintraperitoneally with 0.5 mL of E. muris-infected DH82 cells.

Mice were positively identified as being infected with E. muris via PCRof spleen and blood DNA. DNA was purified from blood and tissue samplesusing a Qiagen QIAamp DNA Mini Kit according to the manufacturer'sinstructions. PCR was performed on a RoboCycler® robotic thermocycler(Stratagene) using 25-μl reactions consisting of 2.5 μl of 10× reactionbuffer (Genscript), 0.2 μl of 100 mM dNTPs (Invitrogen), 1 μl of 10 μMoligonucleotide primer 1 (5′-AGA ACG AAC GCT GGC GGC AAG C-3″) andoligonucleotide primer 2 (5′-CGT ATT ACC GCG GCT GCT GGC A-3′), and 0.2μl of 5 U/μl Taq polymerase (Genscript) in a thermocycling protocolconsisting of a preliminary denaturation step of 94° C. for 5 min,followed by 35 cycles of 94° C. for 1 min, 60° C. for 1 min, and 72° C.for 1 min, followed by a final elongation step of 72° C. for 10 minutes.

Homogenates in fresh growth medium were prepared from spleen samplesobtained from E. muris-infected mice and used in FEA feline embryoniccells. One ml of the E. muris-infected mouse spleen homogenate wasinoculated into uninfected FEA feline embryonic cells in a 25 cm² cellculture flask containing a 24 hour FEA feline embryonic cell monolayer(at ˜80% confluency) and 8 ml of FEA growth medium at a final homogenatedilution of 1:9. Following 5-24 hours incubation at 37° C. with 5% CO2,the culture medium was replaced with 8 ml of fresh FEA Growth Medium.After 5-7 days incubation, the cell monolayer was trypsinized andresuspended in 2 ml fresh FEA growth medium. One ml of this E.muris-infected cell suspension was inoculated into a 75 cm² cell cultureflask containing a 24 hour FEA feline embryonic cell monolayer and 30 mlof FEA growth medium. Cells were incubated for 4-7 days at 37° C. with5% CO₂. Infected cultures were passed 5 times using incubation timesranging from 4-9 days at 37° C. with 5% CO₂. For further passage, 1 to 2ml of infected cells that were trypsinized and resuspended in 4 to 8 mlof growth medium was used to infect additional uninfected FEA felineembryonic cells. Infected cells were inoculated into flasks containing24-hour-old FEA feline embryonic cell monolayer, incubated for 5-48 hrsat 37° C. with 5% CO₂, refed with fresh growth medium, and incubatedfurther for 4-9 days at 37° C. with 5% CO₂. The presence of E. muris inthe cultures of FEA feline embryonic cells was confirmed by the use ofIFA and PCR, as described above.

Example 5 Growth of E. muris on a Homogenous Population of FEF Cells

Homogenates in growth medium were prepared from spleen samples obtainedfrom E. muris-infected mice as described above and was used to infectFEF cells, cultured as described above. Each uninfected cell line wasinoculated with 0.5 ml E. muris-infected spleen homogenate per 25 cm²cell culture flask which contained a 24 hour monolayer and 8 ml growthmedium. This is a final homogenate dilution of 1:17. Following 24 hoursincubation at 37° C. with 5% CO₂, the culture medium was replaced with 8ml of appropriate fresh culture medium. After 7 days incubation, thecell monolayer was trypsinized and the entire infected cell contents ofthe 25 cm² flask was inoculated into a 75 cm² flask containing 30 mlfresh medium (this is a 1:3.75 split). Infected cultures were passed 2times using incubation times ranging from 7-8 days at 37° C. with 5%CO₂. The presence of E. muris in the cultures of cells was confirmed bythe use of IFA and PCR, as described above.

Example 6 Growth of E. muris on a Homogenous Population of FCWF-4 Cells

Homogenates in growth medium were prepared from spleen samples obtainedfrom E. muris-infected mice as described above and was used to infectFCWF-4 cells, cultured as described above. Each uninfected cell line wasinoculated with 0.5 ml E. muris-infected spleen homogenate per 25 cm²cell culture flask which contained a 24 hour monolayer and 8 ml growthmedium. This is a final homogenate dilution of 1:17. Following 24 hoursincubation at 37° C. with 5% CO₂, the culture medium was replaced with 8ml of appropriate fresh culture medium. After 7 days incubation, thecell monolayer was trypsinized and the entire infected cell contents ofthe 25 cm² flask was inoculated into a 75 cm2 flask containing 30 mlfresh medium (this is a 1:3.75 split). Infected cultures were passed 2times using incubation times ranging from 7-8 days at 37° C. with 5%CO₂. The presence of E. muris in the cultures of cells was confirmed bythe use of IFA and PCR, as described above.

Example 7 Infection of FEF Cells with N. risticii-infected P388D1 Cells

Materials for infecting FEF cells with N. risticii from N.risticii-infected P388D1 cells can be prepared as described above, or aspreviously described in Vemulapalli, R. et al., J. Clin. Micro. 33(11):2987-2993 (1995), or as previously described in U.S. Pat. No. 4,759,927,which is herein wholy incorporated by reference.

P388D1 cells (ATCC accession no. CC1-46) infected with the 90-12 strainof N. risticii were added at a multiplicity-of-infection (MOI) of 0.0006to 0.0028 in 850-cm² roller bottles containing monolayers of 3-4-day-oldFEF cells. Prior to infection, the spent culture medium in the rollerbottles were replaced with 300 ml of the culture medium used forinfection. After inoculation at the specified MOI, the roller bottleswere incubated at 37° C. without CO₂. Culture medium tested included butnot limited to D-MEM, MEM Earles, and M6B8; 0 to 5% FBS was used. Theinfected cultures were harvested when the cytopathic effect (CPE)reached 75% to 85%, which ranged from 8-16 days depending on the MOIused. CPE observed included swelling, rounding, and detachment ofinfected cells. The cultures were harvested by tapping the sides ofroller bottles to dislodge the cells into the culture medium. Thepresence of infection was confirmed using a standard IFA protocol oncells that were fixed with 70% acetone and 30% methanol in 96-wellplates with an N. risticii monoclonal antibody and fluorescein-labeledgoat anti-mouse IgG (Bethyl Laboratories, Inc., Montgomery, Tex.).

Example 8 Infection of MA-104 Cells with E. Muris

8.1. Propagation of Uninfected MA-104 Cells

One vial of frozen MA-104 cells (monkey embryo kidney epithelial cells,ATCC accession no. CRL-2378.1) was thawed, clarified, and used toinoculate a 75-cm² cell culture flask containing MA-104 Growth Medium,and incubated at 37° C. with 5% CO₂. MA-104 Growth Medium consists ofEagles Minimal Essential medium with Earles BSS and 2 mM L-glutamine(EMEM) which is supplemented with 1.0 mM sodium pyruvate, 0.1 mMnonessential amino acids, 1.5 g/1 sodium bicarbonate, an additional 1%L-glutamine and 10% FBS. After incubation for 5-7 days, the cultureswere ready for passage when the monolayer was 90-95% confluent. Aftertreatment with 0.25% trypsin, the cells were passed at a split ratio of1:5 to 1:10.

8.2. Preparation of Homogenous Population of E. Muris-Infected MA-104Cells

Homogenates were prepared from spleen samples obtained from E.muris-infected mice and used to infect MA-104 cells. E. muris-infectedmouse spleen homogenate was used to overlay MA-104 cells in 25-cm2 cellculture flasks. Twenty-four hour old MA-104 monolayers at ˜85%confluency were inoculated with a 1:91 dilution of spleen homogenate(0.1 ml spleen homogenate into 9 ml existing 24 hour flask media).

MA-104 Growth Medium consists of Eagles Minimal Essential medium withEarles BSS and 2 mM L-glutamine (EMEM) which is supplemented with 1.0 mMsodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/1 sodiumbicarbonate, an additional 1% L-glutamine and 10% FBS. After 5 days ofincubation at 37° C. with 5% CO₂, the cells were treated with trypsinand all the cells were resuspended in 30 ml of fresh MA-104 GrowthMedium; this 30 ml was then dispensed into a 75 cm² cell culture flask.After 8 days of incubation at 37° C. with 5% CO₂, these cells weretreated with trypsin and all the cells were resuspended in 50 ml offresh MA-104 Growth Medium; this 50 ml was then dispensed into a 175 cm²cell culture flask. For further passage, 3 mLs of infected cells thatwere trypsinized and resuspended in 9-10 ml of growth medium wereinoculated into fresh 50 ml growth medium in 175 cm² cell cultureflasks. Uninfected MA-104 cells were added during passaging when thecytopathic effect in the infected cells appeared advanced, the infectedcells were sparse, or the infected cells appeared weak. The incubationrange for the infected cells was 5-8 days at 37° C. with 5% CO₂. Thepresence of E. muris in the cultured MA-104 cells was confirmed by useof IFA.

* * *

All patents, published patent application and other publications areherein incorporated by reference in their entirety.

We claim:
 1. A method for continuously culturing an Ehrlichia speciescomprising: i) obtaining the Ehrlichia species; ii) obtaining felineembryonic fibroblast cells; iii) infecting the feline embryonicfibroblast cells with the Ehrlichia species; and iv) continuouslyculturing said feline embryonic fibroblast cells in feline embryoniccell growth medium under conditions conducive to propagating the felineembryonic fibroblast cells, thereby continuously culturing the Ehrlichiaspecies.
 2. A method for continuously culturing an Ehrlicia speciescomprising: i) infecting a mammal with the Ehrlicia species; ii)obtaining infected tissue from the infected mammal; iii) contactingfeline embryonic fibroblast cells with the infected tissue; and iv)continuously culturing said feline embryonic fibroblast cells in felineembryonic cell growth medium under conditions conducive to propagatingthe feline embryonic fibroblast cells, thereby continuously culturingthe Ehrlichia species.
 3. The method of claim 2, wherein the infectedtissue from the infected mammal is a spleen homogenate.
 4. A method forcontinuously culturing a Neorickettsia risticii species comprising: i)infecting a mammal with the Neorickettsia risticii species; ii)obtaining infected tissue from the infected mammal; iii) contactingfeline embryonic fibroblast cells with the infected tissue; and iv)continuously culturing said feline embryonic fibroblast cells in felineembryonic cell growth medium under conditions conducive to propagatingthe feline embryonic fibroblast cells, thereby continuously culturingthe Neorickettsia risticii species.
 5. The method of claim 4, whereinthe infected tissue from the infected mammal is a spleen homogenate. 6.A method for continuously culturing Neorickettsia risticii speciescomprising: i) obtaining the Neorickettsia risticii species; ii)obtaining feline embryonic fibroblast cells; iii) infecting the felineembryonic fibroblast cells with the Neorickettsia risticii species; andiv) continuously culturing said feline embryonic fibroblast cells infeline embryonic cell growth medium under conditions conducive topropagating the feline embryonic fibroblast cells, thereby continuouslyculturing the Neorickettsia risticii species.